Immunopathogenesis of Behçet's disease.

Behçet's disease (BD) is a multi-system inflammatory disorder with vasculitic features. It does not suit any of the current pathogenesis-driven disease classifications well, a unifying concept of its pathogenesis is not unanimously conceivable at present, and its etiology is obscure. Still, evidence from immunogenetic and other studies supports the notion of a complex-polygenic disease with robust innate effector responses, reconstitution of regulatory T cells upon successful treatment, and first clues to the role of an, as of yet, underexplored adaptive immune system and its antigen recognition receptors. Without an attempt to be comprehensive, this review aims to collect and organize impactful parts of this evidence in a way that allows the reader to appreciate the work done and define the efforts needed now. The focus is on literature and notions that drove the field into new directions, whether recent or more remote.

Behçet's disease risk-variant HLA-B51/ERAP1-Hap10 alters human CD8 T cell immunity.

OBJECTIVES: The endoplasmic reticulum aminopeptidase (ERAP1) haplotype Hap10 encodes for a variant allotype of the endoplasmic reticulum (ER)-resident peptide-trimming aminopeptidase ERAP1 with low enzymatic activity. This haplotype recessively confers the highest risk for Behçet's diseases (BD) currently known, but only in carriers of HLA-B*51, the classical risk factor for the disease. The mechanistic implications and biological consequences of this epistatic relationship are unknown. Here, we aimed to determine its biological relevance and functional impact. METHODS: We genotyped and immune phenotyped a cohort of 26 untreated Turkish BD subjects and 22 healthy donors, generated CRISPR-Cas9 ERAP1 KOs from HLA-B*51 + LCL, analysed the HLA class I-bound peptidome for peptide length differences and assessed immunogenicity of genome-edited cells in CD8 T cell co-culture systems. RESULTS: Allele frequencies of ERAP1-Hap10 were similar to previous studies. There were frequency shifts between antigen-experienced and naïve CD8 T cell populations of carriers and non-carriers of ERAP1-Hap10 in an HLA-B*51 background. ERAP1 KO cells showed peptidomes with longer peptides above 9mer and significant differences in their ability to stimulate alloreactive CD8 T cells compared with wild-type control cells. CONCLUSIONS: We demonstrate that hypoactive ERAP1 changes immunogenicity to CD8 T cells, mediated by an HLA class I peptidome with undertrimmed peptides. Naïve/effector CD8 T cell shifts in affected carriers provide evidence of the biological relevance of ERAP1-Hap10/HLA-B*51 at the cellular level and point to an HLA-B51-restricted process. Our findings suggest that variant ERAP1-Hap10 partakes in BD pathogenesis by generating HLA-B51-restricted peptides, causing a change in immunodominance of the ensuing CD8 T cell response.

Single-cell cloning of human T-cell lines reveals clonal variation in cell death responses to chemotherapeutics.

Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.

Flow Plex-A tool for unbiased comprehensive flow cytometry data analysis.

INTRODUCTION: The information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training. METHODS: We aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self-generated data sets. RESULTS: We developed Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs "healthy", "old" vs "young", and facilitates the identification of cell populations with potential biologic relevance to states of disease and health. CONCLUSIONS: We provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets.

The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils.

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.